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Clinical features of childhood acute myeloid leukaemia with specific gene rearrangements

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E Frascella
E Frascella
E Frascella
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Roberto Rondelli at Policlinico S.Orsola-Malpighi
Roberto Rondelli
Martina Pigazzi at University of Padova
Martina Pigazzi
Claudia Zampieron at Istituto Zooprofilattico Sperimentale delle Venezie
Claudia Zampieron
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CORRESPONDENCE
Clinical features of childhood acute myeloid leukaemia with specific gene
rearrangements
Leukemia (2004) 18, 1427–1429. doi:10.1038/sj.leu.2403410
Published online 17 June 2004
TO THE EDITOR
Specific gene rearrangements seem to distinguish distinct
subsets of acute myeloid leukaemia (AML) with different
features and prognosis, and some reports suggest that the
epidemiological distribution of AML could vary among coun-
tries.
1,2
To date, cytogenetic examination has been used to study
the frequency of these genetic alterations in a large series of
children
3–5
with AML; nevertheless, in a proportion of cases,
these gene rearrangements may be cryptic and undetectable by
conventional cytogenetic techniques.
6,7
To evaluate the fre-
quency of specific gene rearrangements, the corresponding
clinical morphological features at diagnosis and the potential
prognostic impact on patients’ long-term survival, we screened
by RT-PCR five different chimaeric transcripts (AML1-ETO,
CBFb-MYH11, PML-RARa, MLL-AF9 and BCR-ABL) in a series
of 270 Italian children with AML, treated with AIEOP-LAM 87–
92, BFM 83-93 and AIDA protocols between 1988 and 1998,
and whose RNA were available and morphology had been
centrally reviewed.
Of the cases, 45% were positive for one fusion gene. The
frequency of AML1-ETO, CBFb-MYH11, PML-RARaand MLL-
AF9 were 14, 6.3, 20 and 4%, respectively, while BCR-ABL was
observed in only two cases. The frequency of AML1-ETO, MLL-
AF9 and CBFb-MYH11 found was comparable with those of
t(8;21), t(9;11) and inv(16) reported previously, suggesting that
these gene rearrangements do not have geographic hetero-
geneity. On the contrary, PML-RARa-positive AML represented
20% of our cohort. This is a higher frequency than that reported
in other studies of children from Northern Europe or the United
States, but the increased frequency of promyelocytic leukaemia
in Italian AML children has already been described.
Cytogenetic analysis was successfully carried out in 153
patients, while it failed or was not performed in 117 patients.
Abnormalities were identified in 90/153 (59%), the frequency of
t(8;21), inv(16), t(15;17) and t(9;11) was 9.8, 2, 17 and 2%,
respectively; the 63 remaining cases (41%) had a normal
karyotype. In the t(8;21)-positive AML cases, the most common
associated alterations involved chromosome 9. Among patients
with normal karyotype, RT-PCR identified 17/63 (27%) chimae-
ric transcripts, and six further cases with translocations
undetected by cytogenetics were found among the 43 children
reported to have an abnormal karyotype. Considering t(8;21),
inv(16), t(15;17) and t(9;11) together, 130/153 (85%) patients,
who had a successful analysis were correctly classified
cytogenetically. Then, in our experience, cytogenetics showed
comparable specificity (100%), but lower sensitivity (67%) than
RT-PCR. A general agreement exists in considering RT-PCR
more sensitive than conventional cytogenetics in identifying
specific gene rearrangements and the fusion genes studied have
been described in the absence of identifiable translocations.
Nevertheless, the molecular technique is not capable of
identifying associated abnormalities. Therefore, since the
genetic studies in childhood AML allow for the identification
of patient subgroups who probably can benefit from a more
tailored therapy and the fusion gene identified can represent a
useful target for the minimal residual disease study, ideally all
cases should be studied by RT-PCR and cytogenetics: two
complementary methods in the genetic characterisation of
leukaemia.
Clinical data collected included FAB subtypes, gender, age,
WBC count at diagnosis and extramedullary involvement,
whenever available (Table 1). The low number of M7-AML
was due to the scarce number of cells collected in megakaryo-
blastic leukaemia.
The median age at diagnosis for all patients was 7.8 (range 0–
19.5). Cases with AML1-ETO and PML-RARafusion genes were
older patients (median age 8.2 and 9.5 years, respectively),
while the median age of patients with CBFb-MYH11 and MLL-
AF9 was 6.7 and 3.2 years, respectively.
A strong, although not exclusive, association between M3,
M4 and M5 FAB subgroups and the presence of PML-RARa,
CBFb-MYH11 and MLL-AF9 gene rearrangements, respectively,
was found. The AML1-ETO-positive cases represented 43% of
M2 AML; however, this gene rearrangement was frequently
found in M1 patients as well. As a whole, 92% (34/37) of AML1-
ETO-positive AML were classified as FAB-M1 or FAB-M2.
The median WBC count at diagnosis was significantly higher
in CBFb-MYH11-positive cases, while PML-RARa- and AML1-
ETO-positive cases had the lowest values. Also, among M2 and
M1 plus M2 FAB subgroups this value was significantly lower in
AML1-ETO-positive AML than in negatives (16 000/mlvs 30 800/
ml, P¼0.027; 16 000/mlvs 34 200/ml, P¼0.0005).
Extramedullary disease involved the liver (41%), spleen
(37%), node (15%), CNS (8%) and other sites (9%). The FAB-
M3 subtype and the presence of PML-RARaor AML1-ETO
fusion genes were significantly associated with low frequency of
extramedullary involvement (P¼0.0002, P¼0.002 and
P¼0.0047, respectively), while a correlation with the presence
of extramedullary disease was found with the presence of MLL-
AF9, M4 and M5 FAB subgroups and a WBC count at diagnosis
higher than 20 000/ml(P¼0.024, P¼0.017, P¼0.018 and
P¼0.046, respectively). CNS involvement was found in 19
patients and, in five of them, was the sole extramedullary site
involved. A significant correlation was found only between CNS
disease and a WBC count of X50 000/ml at diagnosis
(P¼0.002).
In our series, the genetic subgroups showed a characteristic
clinical and morphological profile with regard to FAB subtype,
age distribution, WBC count at diagnosis, extramedullary
Received 6 February 2004; accepted 4 May 2004; Published online
17 June 2004
Correspondence: E Frascella, Pediatric Hematology-Oncology,
Department of Pediatrics, University of Padova, via Giustiniani 3,
Padova 35128, Italy;
Fax: þ39 0498211462; E-mail: emanuela.frascella@unipd.it
Leukemia (2004) 18, 1427–1450
&2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00
www.nature.com/leu
disease and CNS involvement. PML-RARa-positive cases
showed a strong association with the FAB-M3 subtype, a
progressive increase of frequency with age, a low WBC count
and only occasional extramedullary and CNS involvement.
AML1-ETO-positive leukaemia demonstrated a peculiar distri-
bution with respect to specific age groups, with the highest
incidence between 5 and 10 years, and a close, but not
exclusive, correlation with the FAB-M2 subtype. Furthermore, a
moderate increase of the WBC count at diagnosis seems to be a
typical feature of t(8;21) AML; indeed, in our patients the
median WBC count was 16 000/ml, similar to values reported by
other authors, while, unlike the results of other studies, we did
not observe an increased frequency of CNS involvement in this
group. CBFb-MYH11 AML were associated with the FAB-M4
subtype, even if occasionally found in M2 and M5 AML, and
were characterized by a high WBC count at diagnosis with
frequent CNS involvement. MLL-AF9-positive AML was the
group with the highest percentage of infants and the lowest
median age; these cases often showed extramedullary disease
and CNS involvement even though the median WBC count at
diagnosis was o20000/ml.
Complete remission was achieved in 84.5% of cases. In
patients with AML positive for AML1-ETO, CBFb-MYH11, MLL-
AF9 and PML-RARa, the CR rate was 91, 87.5, 91, and 84.3%,
respectively. The lowest CR rate was observed in patients less
than p1 year (63%, P¼0.007) and when the WBC count at
diagnosis was higher than 50 000/ml (76.9%, P¼0.029).
Univariate and multivariate survival analyses were carried out
on a series of 200 patients, excluding secondary AML, Down
syndrome and promyelocytic leukaemia since specific proto-
cols, including ATRA, were implemented to treat APL over the
years. Variables considered in univariate analysis were: gender,
extramedullary disease, CNS involvement, presence of AML1-
ETO/CBFb-MYH11 gene rearrangements, FAB subtypes, median
WBC count at diagnosis p20 000/mlor420 000/ml and age p1
year or 41 year. The significant prognostic factors for both EFS
and OS are listed in Table 2. Owing to the scarce number of
cases and the presence of a case with Down syndrome, the MLL-
AF9 gene rearrangement was not considered in the survival
analysis; nevertheless, in our series 54.5% of the patients were
still alive in remission. Multivariate analysis (Table 3) confirmed
that the absence of the AML1-ETO and CBFb-MYH11 gene
rearrangements, a WBC count of 420 000/ml at diagnosis and
an age p1 year are independent unfavourable prognostic
factors. A general consensus exists in considering CBFb-
MYH11-positive AML as a group characterised by better CR
and better survival rates than other AML, in spite of hyperleu-
cocytosis at diagnosis and CNS involvement, while conflicting
Table 1 Presenting clinical and biological features
Cohort WBC 10
3
ml
a
ED
b
(CNS pos)
n % Median
#^
Range % %
Total 270 100 22.8 0.8–495 56.7 (8.0)
Sex
Female 126 47 25.9 1.4–495 56.8 (9.6)
Male 144 53 17.4 0.8–340 56.7 (6.4)
Age (years)
p1 22 8 29.3 2.9–190 60.0 (15.8)
41p5 65 24 25.1 1.8–495 64.6 (6.1)
45p10 85 31 24.5 0.8–425 45.1 (7.0)
410p15 86 32 22.2 1–342 58.7 (9.3)
415 12 4 11.3 1.9–41.2 41.7 (F)
FAB subgroup
M0 12 4.5 32.0 11.7–264 70.0 (10.0)
M1 39 14.5 44.7 1–400 63.0 (7.4)
M2 58 21.5 21.6 1.4–322 50.0 (7.7)
M3 54 20 5.6
#
0.8–290 34.0 (2.0)
M4 43 16 48.7
^
3–425 70.7 (15.0)
M5 57 21 29.9 1.8–495 71.1 (9.6)
M6 4 1.5 NE NE NE
M7 3 1 NE NE NE
Gene rearrangement
AML1-ETO 37 14 16.0
^
1.5–329 39.3 (7.1)
CBFb-MYH11 17 6.3 51.4
#
11.2–220 60.0 (14.3)
PML-RARa 54 20 5.6
#
0.8–290 38.2 (2.0)
MLL-AF9 11 4 16.7 1.8–400 91.0 (18.0)
BCR-ABL 2 0.7 NE NE NE
Negative 149 55 32.2 1.0–495 65.3 (6.3)
ED ¼extramedullary disease; NE ¼not evaluable.
a
Percentages calculated on 252 pts. for whom data were available.
b
Percentages calculated on 236 pts. for whom data were available.
c
Median sign test significant:
^
Po0.05 and
#
Po¼0.001. Control
group was total population.
Table 2 Univariate survival analysis: significant variables
Variable Pts. Overall survival Event-free survival
4 years % value (s.e.) Log-rank P-value 4 years % value (s.e.) Log-rank P-vaue
All patients 200 45.3 (3.6) F41.0 (3.6) F
AML1ETO+CBFb-MYH11 positive 52 58.4 (7.0) 0.024 54.7 (7.3) 0.029
AML1ETO+CBFb-MYH11 negative 148 41.0 (4.2) 36.4 (4.0)
WBC p20000/ml 74 59.5 (5.9) 0.0038 53.5 (6.0) 0.0038
WBC 420000/ml 116 37.4 (4.6) 33.8 (4.5)
Age p1 year 19 25.3 (10.2) 0.0058 21.0 (9.3) 0.002
Age 41 year 181 47.5 (3.8) 43.2 (3.8)
Table 3 Multivariate analysis by Cox’s model
Variable Overall survival Event-free survival
Hazard
ratio
95% hazard ratio
confidence limits
P-value Hazard
ratio
95% hazard ratio
confidence limits
P-value
AML1-ETO and CBFb-MYH11 neg. 1760 1.056–2.933 0.030 1662 1.012–2.732 0.045
WBC 420 000/ml 1793 1.181–2.721 0.006 1725 1.148–2.592 0.009
Age p1 year 1860 1.035–3.344 0.038 1941 1.101–3.422 0.022
Correspondence
1428
Leukemia
data exist with regard to AML1-ETO-positive AML. In these
genetic subgroups, an improvement of the outcome in patients
treated with high-dose cytarabine has been described.
8
There-
fore, the poor outcome occasionally reported might be related to
the absence of high-dose cytarabine in the treatment regimens.
In our series, many patients received high-dose cytarabine and,
thus, the favourable outcome observed in patients with AML1-
ETO- and CBFb-MYH11-positive AML could depend on the
treatment received.
Finally, we performed long-term survival analysis to evaluate
the events time distribution considering that the main cause of
failure in AML was relapse (Figure 1). A time interval from
diagnosis of 18 months or longer was significantly more frequent
among AML lacking AML1-ETO and CBFb-MYH11 transcripts
than in positive cases (12.3 vs 2%, respectively, P¼0.02). As a
consequence, the survival probability showed a progressive
decrease in the first group of patients so that their corresponding
10-year survival probability was 31.774.8%. On the contrary,
patients with AML1-ETO- and CBFb-MYH11-positive AML
achieved a plateau at 4 years for both EFS and OS. Among
infants, all events occurred within 14 months from diagnosis and
were mainly due to induction death. Thus, the presence of early
relapses seems to distinguish the AML1-ETO/CBFb-MYH11-
positive cases from the negative ones. These data have been not
previously reported and suggest that the molecular study of
minimal residual disease, performed during the first 2 years from
diagnosis, could allow for the timely identification of early
events in AML with rearrangement of the core binding factor
genes AML1 and CBFb, since relapses are the main cause of
failure in these patients. In addition our study suggests that a
long-term follow-up could be necessary to evaluate the true
probability of survival in patients at high risk of late relapse.
Acknowledgements
We thank Professor G Valsecchi for her helpful suggestions, Ms S
Disaro
`for excellent technical assistance, Dr A Leszl for
cytogenetic data and Dr C Case for linguistic consultancy. We
are grateful to all the clinicians of the participating AIEOP centres
for clinical data. We also thank S Fenu, L Sainati, A Cantu`
Rajnoldi and G Basso for the morphological review. This research
was supported by Fondazione Citta
`della Speranza and and
Prog.Fin. 2001/01/X/000177 Ministero Salute OBG, MIUR 60-
40% and CNR Progetto Oncologia.
E Frascella
1
R Rondelli
2
M Pigazzi
1
C Zampieron
1
F Fagioli
3
C Favre
4
AA Lippi
5
F Locatelli
6
M Luciani
7
G Menna
8
C Micalizzi
9
C Rizzari
10
AM Testi
11
A Pession
2
G Basso
1
1
Pediatric Hematology-Oncology Units,
University of Padova, Italy;
2
Pediatric Hematology-Oncology Units,
University of Bologna, Italy;
3
Pediatric Hematology-Oncology Units,
University of Torino, Italy;
4
Pediatric Hematology-Oncology Units,
University of Pisa, Italy;
5
Pediatric Hematology-Oncology Units,
University of Firenze, Italy;
6
Pediatric Hematology-Oncology Units,
University of Pavia, Italy;
7
Pediatric Hematology-Oncology Units,
Bambino-Gesu` Hospital, Roma, Italy;
8
Department of Oncology A.O.R.N. Santobono-
Pausilipon, Napoli, Italy;
9
Hematology-Oncology Units, Gaslini Institute,
Genova, Italy;
10
Pediatric Hematology-Oncology Units,
University of Monza, Italy; and
11
Department of Hematology, University of
Roma, Roma, Italy
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Figure 1 Event-free survival. Patients by molecular markers.
Correspondence
1429
Leukemia
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... The frequency of PML-RARA among Iraqi childhood AML cases was nearly 18 %, which is higher than the results reported from many Western countries [4,28,32,33]. However, the Iraqi incidence was comparable to those in Latin American countries as well as Italy, with an increased incidence of APL (13.6-28.5 %), reaching up to 34 % in Costa Rica [34][35][36]. A possible explanation for this is exposure to environmental factors and/or genetic predisposition related to ethnicity [34,35]. ...
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  • ANN HEMATOL
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... The t(9;11)(p22;q23) MLL-AF9 translocation is the most frequent rearrangement in AML subtype M5a, that represents approximately 15% of AML in children >2 years old and approximately 50% of AML in younger children (8). t(9;11) (p22;q23) MLL-AF9 leukemia has a more favorable prognosis than t(4;11)(q21;q23) MLL-AF4 leukemia (11), but the event-free and overall survival rates in AMLs vary largely depending on the MLL fusion partner gene involved, with t(9;11)(p22;q23) MLL-AF9 having the most favorable outcome and t(6;11)(q27;q23) MLL-AF6 the poorest (3). ...
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BAG1: The Guardian of Anti-Apoptotic Proteins in Acute Myeloid Leukemia
Article
Full-text available
BCL2 associated Athano-Gene 1 (BAG1) is a multifunctional protein that has been described to be involved in different cell processes linked to cell survival. It has been reported as deregulated in diverse cancer types. Here, BAG1 protein was found highly expressed in children with acute myeloid leukemia at diagnosis, and in a cohort of leukemic cell lines. A silencing approach was used for determining BAG1's role in AML, finding that its down-regulation decreased expression of BCL2, BCL-XL, MCL1, and phospho-ERK1/2, all proteins able to sustain leukemia, without affecting the pro-apoptotic protein BAX. BAG1 down-regulation was also found to increase expression of BAG3, whose similar activity was able to compensate the loss of function of BAG1. BAG1/BAG3 co-silencing caused an enhanced cell predisposition to death in cell lines and also in primary AML cultures, affecting the same proteins. Cell death was CASPASE-3 dependent, was accompanied by PARP cleavage and documented by an increased release of pro-apoptotic molecules Smac/DIABLO and Cytochrome c. BAG1 was found to directly maintain BCL2 and to protect MCL1 from proteasomal degradation by controlling USP9X expression, which appeared to be its novel target. Finally, BAG1 was found able to affect leukemia cell fate by influencing the expression of anti-apoptotic proteins crucial for AML maintenance.
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Predictive factors of relapse and survival in childhood acute myeloid leukemia: Role of minimal residual disease
Article
Minimal residual disease (MRD) in acute myeloid leukemia (AML) can be measured either by flow cytometry to detect leukemic immunophenotypes or by PCR amplification of fusion transcripts, gene mutations and overexpressed genes. Flow cytometry MRD is widely applicable but has an intermediate sensitivity; PCR MRD is highly sensitive but has a restricted applicability. MRD, measured at different time points throughout AML treatment, has proven to powerfully predict the risk of relapse. Acute promyelocytic leukemia, a subtype of AML, represents an excellent example of systematic evaluation and established clinical application of MRD performed by PCR. However, a standardization of MRD techniques, threshold levels, time-points and clinical interventions based on its results is still needed in this field. This article is focused on the current available information on MRD as a prognostic marker in childhood AML.
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Nucleophosmin mutations in childhood acute myelogenous leukemia with normal karyotype
Article
Full-text available
  • Sep 2005
Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein involved in leukemia-associated chromosomal translocations, and it regulates the alternate reading frame (ARF)-p53 tumor-suppressor pathway. Recently, it has been demonstrated that mutations of the NPM1 gene alter the protein at its C-terminal, causing its cytoplasmic localization. Cytoplasmic NPM was detected in 35% of adult patients with primary non-French-American-British (FAB) classification M3 acute myeloid leukemia (AML), associated mainly with normal karyotype. We evaluated the prevalence of the NPM1 gene mutation in non-M3 childhood AML patients enrolled in the ongoing Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP-AML02) protocol in Italy. NPM1 mutations were found in 7 (6.5%) of 107 successfully analyzed patients. NPM1-mutated patients carried a normal karyotype (7/26, 27.1%) and were older in age. Thus, the NPM1 mutation is a frequent abnormality in AML patients without known genetic marker; the mutation may represent a new target to monitor minimal residual disease in AML and a potential candidate for alternative and targeted treatments.
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The Importance of Diagnostic Cytogenetics on Outcome in AML: Analysis of 1,612 Patients Entered Into the MRC AML 10 Trial
Article
Full-text available
  • Oct 1998
Cytogenetics is considered one of the most valuable prognostic determinants in acute myeloid leukemia (AML). However, many studies on which this assertion is based were limited by relatively small sample sizes or varying treatment approach, leading to conflicting data regarding the prognostic implications of specific cytogenetic abnormalities. The Medical Research Council (MRC) AML 10 trial, which included children and adults up to 55 years of age, not only affords the opportunity to determine the independent prognostic significance of pretreatment cytogenetics in the context of large patient groups receiving comparable therapy, but also to address their impact on the outcome of subsequent transplantation procedures performed in first complete remission (CR). On the basis of response to induction treatment, relapse risk, and overall survival, three prognostic groups could be defined by cytogenetic abnormalities detected at presentation in comparison with the outcome of patients with normal karyotype. AML associated with t(8;21), t(15;17) or inv(16) predicted a relatively favorable outcome. Whereas in patients lacking these favorable changes, the presence of a complex karyotype, −5, del(5q), −7, or abnormalities of 3q defined a group with relatively poor prognosis. The remaining group of patients including those with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other miscellaneous structural or numerical defects not encompassed by the favorable or adverse risk groups were found to have an intermediate prognosis. The presence of additional cytogenetic abnormalities did not modify the outcome of patients with favorable cytogenetics. Subgroup analysis demonstrated that the three cytogenetically defined prognostic groups retained their predictive value in the context of secondary as well as de novo AML, within the pediatric age group and furthermore were found to be a key determinant of outcome from autologous or allogeneic bone marrow transplantation (BMT) in first CR. This study highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the framework for a stratified treatment approach of this disease, which has been adopted in the current MRC AML 12 trial.
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The Importance of Diagnostic Cytogenetics on Outcome in AML: Analysis of 1,612 Patients Entered Into the MRC AML 10 Trial
Article
Full-text available
  • Nov 1998
Cytogenetics is considered one of the most valuable prognostic determinants in acute myeloid leukemia (AML). However, many studies on which this assertion is based were limited by relatively small sample sizes or varying treatment approach, leading to conflicting data regarding the prognostic implications of specific cytogenetic abnormalities. The Medical Research Council (MRC) AML 10 trial, which included children and adults up to 55 years of age, not only affords the opportunity to determine the independent prognostic significance of pretreatment cytogenetics in the context of large patient groups receiving comparable therapy, but also to address their impact on the outcome of subsequent transplantation procedures performed in first complete remission (CR). On the basis of response to induction treatment, relapse risk, and overall survival, three prognostic groups could be defined by cytogenetic abnormalities detected at presentation in comparison with the outcome of patients with normal karyotype. AML associated with t(8;21), t(15;17) or inv(16) predicted a relatively favorable outcome. Whereas in patients lacking these favorable changes, the presence of a complex karyotype, -5, del(5q), -7, or abnormalities of 3q defined a group with relatively poor prognosis. The remaining group of patients including those with 11q23 abnormalities, +8, +21, +22, del(9q), del(7q) or other miscellaneous structural or numerical defects not encompassed by the favorable or adverse risk groups were found to have an intermediate prognosis. The presence of additional cytogenetic abnormalities did not modify the outcome of patients with favorable cytogenetics. Subgroup analysis demonstrated that the three cytogenetically defined prognostic groups retained their predictive value in the context of secondary as well as de novo AML, within the pediatric age group and furthermore were found to be a key determinant of outcome from autologous or allogeneic bone marrow transplantation (BMT) in first CR. This study highlights the importance of diagnostic cytogenetics as an independent prognostic factor in AML, providing the framework for a stratified treatment approach of this disease, which has been adopted in the current MRC AML 12 trial.
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Chromosomal abnormalities in 478 children with acute myeloid leukemia: Clinical characteristics and treatment outcome in a Cooperative Pediatric Oncology Group study - POG 8821
Article
Full-text available
  • Jan 2000
We determined the type and frequency of chromosomal aberrations in leukemic cells of 478 children diagnosed with acute myeloid leukemia and enrolled in the Pediatric Oncology Group study 8821. Of the 478 cases, 109 (22.8%) had normal karyotypes. Chromosomal abnormalities of 280 patients (58.6%) were classified into subgroups: 11q23 abnormalities (n = 88, 18.4%), t(8;21) (n = 56, 11.7%), t(15;17) (n = 55, 11.5%), inv(16)/t(16;16) (n = 28, 5.9%), trisomy 8 alone (n = 10, 2.1%), monosomy 7 (n = 9, 1.9%), non-Down-associated trisomy 21 alone (n = 7, 1.5%), and rare recurrent chromosomal translocations (n = 27, 5.6%). The remaining 89 patients (18.6%) had miscellaneous clonal abnormalities. Overall, 84.9% of the children achieved a complete remission; the 4-year event-free survival (EFS) estimate was 33.8% +/- 2.4%. Remission rates were significantly higher (96.4%, P =.011) for patients with t(8;21) and inv(16)/t(16;16) but significantly lower (74.5%, P =.022) for those with t(15;17). The 4-year survival rate for all patients was 43.5% +/- 2.4%; for those with an inv(16)/t(16;16), 75.0% +/- 8.6%; a normal karyotype, 53.8% +/- 4.9%; a t(8;21), 51.6% +/- 7.3%; a t(15;17), 39.8% +/- 6.9%; and an 11q23 abnormality, 32.9% +/- 5.1%. Four-year EFS estimates for patients with inv(16)/t(16;16) (58.2% +/- 10.9%, P =.007), t(8;21) (45.1% +/- 7.7%, P =.014), or normal karyotypes (43.1% +/- 5.0%, P =. 012) were higher than the 4-year EFS estimate for all patients, but EFS estimates for patients with t(15;17) (19.6% +/- 8.0%, P =.033) or 11q23 abnormalities (23.8% +/- 4.8%, P =.0013) were lower. EFS estimates did not differ significantly among 11q23 subgroups. Limited analysis suggested that patients with inv(16) can be salvaged better following relapse than those with t(8;21). Thus, patients with an inv(16)/t(16;16) may have high survival rates when treated with chemotherapy alone.
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Cytogenetically cryptic AML1 - ETO and CBF?? - MYH11 gene rearrangements: Incidence in 412 cases of acute myeloid leukaemia
Article
  • Dec 2000
The rearrangements t(8;21)(q22;22) and inv(16)(p13q22) are two of the most frequently seen in acute myeloid leukaemia (AML), accounting for 8% and 4% of cases respectively. Detection of these abnormalities is important for disease management as both are associated with good responses to conventional chemotherapy and prolonged disease-free survival. Recent reports using reverse transcriptase polymerase chain reaction (RT-PCR) suggest that significant proportions of AML cases without a visible t(8;21) or inv(16) show expression of an abnormal fusion gene transcript and, consequently, they could not be detected using conventional cytogenetic analysis alone. We present here a four centre study involving 412 cases of AML screened using both standard cytogenetics and RT-PCR for AML1–ETO and CBFβ–MYH11. We detected a cytogenetic t(8;21) in 31 out of 412 (7·5%) cases and an inv(16) or t(16;16) variant in 27 out of 412 (6·6%) cases. RT-PCR detected only two cases (0·5%) of cryptic t(8;21) and no instances of cryptic inv(16). Both cryptic t(8;21) cases had the classic M2 FAB morphology for this type of disease. Our data concur with the established FAB type distribution of the rearrangements and indicate that cryptic t(8;21) and inv(16) may be much less frequent than reported elsewhere.
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Detection of karyotypic aberrations in acute myeloblastic leukaemia: a prospective comparison between PCR/FISH and standard cytogenetics in 140 patients with de novo AML
Article
In 140 patients with de novo acute myeloid leukaemia (AML) standard cytogenetics were compared with RT-PCR for the detection of t(8;21), t(15;17) and inv(16) and fluorescence in situ hybridization (FISH) for numerical aberrations of chromosomes 7, 8, X and Y. RT-PCR detected 18 cases with t(8;21), 12 with t(15;17) and seven with inv(16). In two cases with t(8;21), two with t(15;17) and four with inv(16) these aberrations had not been detected by standard cytogenetics. There were no false negative PCR results. In 12 patients with these chromosomal changes, standard cytogenetics revealed additional chromosomal aberrations. In 16 patients sole numerical aberrations of the chromosomes 7, 8, X or Y were found by FISH. In these patients the sensitivity of FISH and standard cytogenetics was comparable. In 87 patients no aberrations could be found by PCR and FISH whereas in 24 of these patients standard cytogenetics revealed an abnormal karyotype. These data recommend the combination of standard cytogenetics and molecular techniques to improve the sensitivity for the detection of genetic lesions in AML. Once chromosomal markers have been identified by combined analysis these markers could be used to monitor residual disease during/after chemotherapy, by RT-PCR and/or FISH.
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Remote Raman Spectroscopy for Process Control
Article
  • May 1995
  • VIB SPECTROSC
The purpose of this paper is to describe the Raman tool presently used for the control of a C8 aromatics separation process based upon simulated moving-bed chromatography. The spectrometer and the laser are remote and connected via 30-m optical fibres with the sensors located at four different points at the process. These sensors operate through sapphire windows without generation of the Raman spectrum of the optical fibres. The Raman spectra are recorded by using a CCD detector which has a capability of 12 simultaneous recordings. The relative concentration of six components at four points of the unit is delivered every 15 s through a quantification software which takes into account the temperature of the fluid. The procedure of calibration based upon a classical analysis of the spectra is described in detail. The frequency domain is restricted to 650–930 cm−1 in order to provide a quick reading of the CCD detector. The quantification uncertainty with reference to chromatographic analyses is ±0.5% for all values in the 0–100% relative concentration range and 25–180°C temperature range with a low fluorescence level. The influence of the fluorescence intensity on the quality of the results is discussed. The efficiency of this Raman device is illustrated on the ELUXYL® pilot unit installed in the IFP centre of Solaize. The real-time drawings of the internal concentration profiles, the purity of para-xylene and the loss of para-xylene in the raffinate stream allow to control the unit.
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Clinical and prognostic significance of chromosomal abnormalities in childhood acute myeloid leukemia de novo
Article
  • Feb 1995
We report on the chromosomal pattern of 120 patients with childhood AML de novo. One hundred and fifteen patients (96%) had adequate samples for analysis; 98 (85%) of these showed clonal karyotypic abnormalities. They were classified into cytogenetic subgroups which were closely correlated with FAB subtypes: t(8;21) and M2 (n = 9); t(15;17) and M3 (n = 12); inv(16) and M4Eo (n = 9); t(9;11) and M5a (n = 10); t(11q23) other than t(9;11) and M4-M5 (n = 11); and t(1;22) and M7 (n = 4). In patients with -7/del(7q) (n = 6), leukemia was preceded by MDS in half of the cases, although they had diverse FAB subtypes. Thirty-seven patients had miscellaneous abnormalities. Despite a high CR rate, patients with t(8;21) had a very poor survival: only one child was event-free at 3 years from diagnosis. One third of patients with t(15;17) died during induction. Those eight who achieved CR fared well: only two relapsed, and six were event-free survivors. Patients with inv(16) had a high remission rate and a long survival: five children were in CR 20 to 136 months. Both groups with t(9;11) and t(11q23) had a high remission rate: however, outcome was superior for the t(9;11) group when compared to either the t(11q23) group (EFS at 3 years +/- SE, 56 +/- 17% vs. 11 +/- 10%, p = 0.07) or to the remaining patients (p = 0.06). Both -7/del(7q) and t(1;22) groups had low CR rates (50%) and poor survival. Cytogenetic analysis identifies clinically distinct subsets of childhood AML and is useful in tailoring treatment for these patients. Favorable cytogenetic groups (t(15;17), inv(16), and t(9;11)) may do well with current therapy protocols, whereas unfavorable groups (t(11q23), t(8;21), -7/del(7q), and t(1;22)) require more effective therapies.
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Acute promyelocytic leukemia in children: Experience of the Italian Pediatric Hematology and Oncology Group (AIEOP)
Article
  • Sep 1994
Scanty information is available on acute promyelocytic leukemia (APL) in children, and whether differences are present with respect to the adult form. The experience of the Italian Pediatric Hematology and Oncology Group (AIEOP) will be presented with respect to the following aspects: 1. Incidence of APL. The incidence of APL is generally considered to account for 3-9% of acute myelogenous leukemia (AML) in children and approximately 10-15% in adults. Recently a single Italian pediatric institution reported that APL constituted one third of observed acute nonlymphocytic leukemia (AnLL) cases. Data from the AIEOP cooperative study group have confirmed that APL in Italy is more frequently observed in children as compared to other countries. Environmental and/or genetic factors should be considered to explain such differences. 2. Diagnosis of M3v. The clinical and biological features of the largest series of childhood M3v will be presented and the problems encountered in the proper separation of 'classic' M3 and M3v in children will be discussed. 3. Clinical Aspects. The clinical features of the APL patients enrolled in the AIEOP study groups since 1989, will be presented with emphasis on the recent experience with the use of all-trans retinoic acid. 4. Analysis of PML/RAR alpha Fusion Transcripts. An RT-PCR analysis of 32 pediatric APL cases from cryopreserved bone marrow samples has been performed. It is concluded that APL in children did not differ significantly from the adult form, with the exception of a higher incidence of PML bcr3 breakpoint.
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Detection of karyotypic aberrations in acute myeloblastic leukaemia: a prospective comparison between PCR/FISH and standard cytogenetics in 140 patients with de novo AML
Article
  • Nov 1998
In 140 patients with de novo acute myeloid leukaemia (AML) standard cytogenetics were compared with RT-PCR for the detection of t(8;21), t(15;17) and inv(16) and fluorescence in situ hybridization (FISH) for numerical aberrations of chromosomes 7, 8, X and Y. RT-PCR detected 18 cases with t(8;21), 12 with t(15;17) and seven with inv(16). In two cases with t(8;21), two with t(15;17) and four with inv(16) these aberrations had not been detected by standard cytogenetics. There were no false negative PCR results. In 12 patients with these chromosomal changes, standard cytogenetics revealed additional chromosomal aberrations. In 16 patients sole numerical aberrations of the chromosomes 7, 8, X or Y were found by FISH. In these patients the sensitivity of FISH and standard cytogenetics was comparable. In 87 patients no aberrations could be found by PCR and FISH whereas in 24 of these patients standard cytogenetics revealed an abnormal karyotype. These data recommend the combination of standard cytogenetics and molecular techniques to improve the sensitivity for the detection of genetic lesions in AML. Once chromosomal markers have been identified by combined analysis these markers could be used to monitor residual disease during/after chemotherapy, by RT-PCR and/or FISH.
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Patients With t(8;21)(q22;q22) and Acute Myeloid Leukemia Have Superior Failure-Free and Overall Survival When Repetitive Cycles of High-Dose Cytarabine Are Administered
Article
  • Dec 1999
To examine the effect of single compared with repetitive (at least three) cycles of high-dose cytarabine after induction therapy for patients with acute myeloid leukemia (AML) who have the t(8;21)(q22;q22) karyotype. Patients entered onto the study had AML and t(8;21) and attained a complete remission on four successive Cancer and Leukemia Group B studies. In these studies, either > or = three cycles of high-dose cytarabine or one cycle of high-dose cytarabine was administered, followed by sequential cyclophosphamide/etoposide and mitoxantrone/diaziquone with or without filgrastim support. Outcomes of these two groups of t(8;21) patients were compared. A total of 50 patients with centrally reviewed AML and t(8;21) were assigned to receive one (n = 29) or > or = three cycles (n = 21) of high-dose cytarabine as postinduction therapy. The clinical features of these two groups of patients were similar. Initial remission duration for t(8;21) patients assigned to one cycle of high-dose cytarabine was significantly inferior (P =.03), with 62% of patients experiencing relapse with a median failure-free survival of 10.5 months, compared with the group of patients who received > or = three cycles, in which only 19% experienced relapse and failure-free survival is estimated to be greater than 35 months. Furthermore, overall survival was also significantly compromised (P =.04) in patients assigned to one cycle of high-dose cytarabine, with 59% having died as a consequence of AML, compared with 24% of those who received > or = three cycles of high-dose cytarabine. These data demonstrate that failure-free survival and overall survival of patients with t(8;21)(q22;q22) may be compromised by treatment approaches that do not include sequential high-dose cytarabine therapy.
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